Molecular tools for differentiating normal breast tissue and cells
from cancerous breast tissue and cells are provided. The tools are
derived from a novel tumor suppressor gene which encodes a protein
referred to hereinafter as the "EDG1" protein. One tool
is an isolated polynucleotide which encodes the EDG1 protein. The
other tool is an antibody which is immunospecific for the EDG1 protein.
Methods of detecting cancerous cells which employ the antibody and
polynucleotide are also provided. Methods for decreasing proliferation
of breast cancer cells, prostate cancer cells, testicular cancer
cells, and ovarian cancer cells are also provided. Such method comprises
increasing levels of the EDG1 protein in such cells
What is claimed is:
1. An isolated polynucleotide comprising a sequence selected from
the group consisting of: (a) a nucleic acid sequence of at least
200 nucleotides which is a portion of SEQ ID NO:1 or the complement
thereof; and, (b) a nucleic acid sequence of at least 200 nucleotides
which hybridizes to SEQ ID NO:1 or the complement thereof, under
2. The isolated polynucleotide of claim 1 wherein said nucleic
acid sequence hybridizes to SEQ ID. NO. 2 or the complement thereof
under highly stringent conditions.
3. An isolated polynucleotide comprising a sequence which encodes
EDG1 protein or a functional equivalent thereof, wherein said EDG1
protein comprises SEQ ID NO. 2, and wherein said functional equivalent
comprises a sequence which is at least 85% identical to SEQ ID NO.
4. The isolated polynucleotide of claim 3 wherein the functional
equivalent is immunologically cross reactive with an antibody raised
using said EDG1 protein as an immunogen.
5. The isolatred polynucleotide of claim 3 wherein the functional
equivalent inhibits proliferation of MCF-7 cells.
6. The isolated polynucleotide of claim 3 wherein said polynucleotide
comprises part of an expression vector, a viral genome, or a liposome.
7. An isolated EDG1 protein or a protein which is functional equivalent
said EDG1 protein, wherein said EDG1 protein comprises SEQ ID NO.
2, and wherein said functional equivalent comprises a sequence which
is at least 85% identical to SEQ ID NO. 2.
8. The isolated protein of claim 7 wherein said protein is a functional
equivalent of said EDG1 protein and is immunologically cross reactive
with an antibody raised using said EDG1 protein as an immunogen.
9. The isolated protein of claim 7 wherein said protein is a functional
equivalent of said EDG1 protein and inhibits proliferation of MCF7
10. The isolated protein of claim 7 wherein said protein is a fusion
protein and comprises a tag for labeling or isolating said protein.
11. A polypeptide which comprises a contiguous sequence within
SEQ ID NO. 2, wherein said contiguous sequence is at least 8 amino
acids in length, and wherein said polypeptide is a functional equivalent
of human EDG1 protein.
12. The polypeptide of claim 11 wherein said polypeptide is immunologically
cross-reactive with an antibody raised using EDG1 protein as an
13. The polypeptide of claim 11 wherein said polypeptide inhibits
proliferation of MCF-7 cells.
14. The polypeptide of claim 10, wherein said polypeptide comprises
SEQ ID NO. 3.
15. An antibody which binds to one or more epitopes in human EDG1
protein, wherein said EDG1 protein comprises SEQ ID NO. 2.
16. The antibody of claim 15 wherein said antibody is a monoclonal
17. A method of detecting cancerous cells in a biological test
sample obtained from a subject known to have or suspected of having
a cancer selected from the group consisting of breast cancer, testicular
cancer, prostate cancer, uterine cancer, cervical cancer, ovarian
cancer, and colon cancer, comprising: a) contacting the test sample
with anti-EDG1 antibody under conditions wherein binding of said
antibody to EDG1 protein occurs; and b) assaying for a complex between
the antibody and a protein in the test sample, wherein a decrease
in the level of the antigen-antibody complex in the test sample,
as compared to the level of the antigen-antibody complex in a control
sample, indicates that the test sample contains or was derived from
18. The method of claim 17 wherein the test sample is a tissue
sample or cell sample, and wherein said test sample is assayed by
an immunocytochemical procedure which permits a determination of
the intracellular location of the antigen-antibody complex.
19. A method of detecting cancerous cells in a biological test
sample obtained from a subject known to have or suspected of having
a cancer selected from the group consisting of breast cancer, testicular
cancer, prostate cancer, uterine cancer, cervical cancer, ovarian
cancer, and colon cancer, comprising: assaying for EDG1 transcript
in said test sample, wherein a decrease in the level of said EDG1
transcript in said test sample, as compared to the level of said
EDG1 in a corresponding control sample, indicates that the test
sample contains or was derived from cancerous cells.
20. The method of claim 19 wherein said sample is assayed by contacting
said sample with a polynucleotide which is complementary to a contiguous
sequence in SEQ ID NO.1 under stringent hybridization conditions.
21. The method of claim 19 wherein said sample is assayed by a
reverse-transcriptase polymerase chain reaction which employs a
primer derived from SEQ ID NO. 1.
22. A method for decreasing proliferation of cancer cells selected
from the group consisting of a breast cancer cells, prostate cancer
cells, testicular cancer cells, ovarian cancer cells, uterine cancer
cells, cervical cancer cells, and colon cancer cells, said method
comprising increasing EDG1 protein activity in said cells.
23. The method of claim 22 wherein levels of EDG1 protein activity
in said cells is increased by contacting the cells with EDG1 protein,
a functional equivalent of EDG1 protein, or a biologically active
fragment of EDG1 protein under conditions which permit uptake of
said protein, said functional equivalent or said biologically active
24. The method of claim 22 wherein EDG1 protein activity is increased
in said cells by contacting said cells with a nucleic acid comprising:
i) a sequence encoding EDG1 protein, a functional equivalent of
EDG1 protein, or a biologically active fragment of EDG1 protein,
and ii) a promoter active in the cancer cell, wherein the promoter
is operably linked to the sequence encoding EDG1 protein, a functional
equivalent of EDG1 protein, or a biologically active fragment of
EDG1 protein, respectively, under condition permitting uptake of
said nucleic acid by the cancer cell.
25. A primer set for amplifying an EDG1 transcript, said primer
set comprising a first primer comprising a sequence which is identical
to a first contiguous sequence in SEQ ID NO.1, and a second primer
comprising a sequence which is complementary to a second contiguous
sequence in SEQ ID NO. 2, wherein said second contiguous sequence
is downstream of said first contiguous sequence.
26. The primer set of claim 25 wherein said first primer and said
second primer each are at least 12 nucleotides in length.
CROSS REFERENCE TO RELATED APPLICATIONS
 This invention claims priority to United States Provisional
Patent Application Serial No.: 60/,238,187 filed Oct. 5, 2000.
 Breast cancer is a significant health problem for women
in the United States and throughout the world. Despite recent advances
in detection and treatment of the disease, breast cancer remains
the second leading cause of cancer-related deaths in women. Management
of the disease currently relies on a combination of early diagnosis
through routine breast screening procedures and aggressive treatment.
Such treatment may include surgery, radiotherapy, chemotherapy,
hormone therapy or combinations of these therapies.
 Ninety-five percent of all breast tumors, at least initially,
are dependent on estrogens for growth. Estrogens are steroid hormones
that are essential for normal sexual development and functioning
of female reproductive organs. Estrogens are also important for
growth, differentiation, and functioning of the testis, epididymis
and prostate in males. Estrogens also have important non-reproductive
effects on bones and the heart. Estrogens comprise a group of natural
and synthetic substances. Natural estrogens include estradiol (i.e.,
17-.beta.-estradiol or E2), estrone and estriol. Estrogens are sometimes
given therapeutically in the form of a conjugate, such as for example,
ethinyl estradiol, conjugated estrogens or diethylstilbestrol.
 Tissues in the body that are responsive to estrogens are
called "estrogen-sensitive" or "estrogen-responsive"
tissues and include cells of the urogenital tract, cardiovascular
system and skeletal system. The cells that comprise estrogen-sensitive
tissues contain estrogen receptors (ER). ER can be of the .alpha.
type or .beta. type. Estrogens enter cells and bind to ER in the
cytoplasm of such cells and an estrogen-ER complex is formed. Herein,
a molecule such as estrogen that binds to a receptor is generally
called a "ligand." Herein, a receptor such as ER that
has formed a complex with a ligand is called a "liganded"
 Once the estrogen ligand binds to ER, the estrogen-ER complex
migrates to the nucleus of the cell and binds to specific sequences
of DNA within the cellular genome called "estrogen response
elements." Such estrogen response elements are located in the
promoters of specific genes in the cell nucleus.
 Binding of the estrogen-ER complex to estrogen-responsive
elements causes activation or suppression of the transcription of
the specific genes (Beato, et al., 1995, Cell, 83:851-7.; Katzenellenbogen,
et al., 1995, J Steroid Biochem Mol Biol, 53:387-93.; Tsai and O'Malley,
1994, Annu Rev Biochem, 63:451-86.). The activation or suppression
of specific gene transcription is one type of molecular and/or cellular
response that can result from formation of a ligand-receptor complex.
When such a response occurs, the receptor is said to have been "activated."
 Estrogen-ER complexes, therefore, act as transcription factors
to regulate the expression of these genes. When a ligand binds to
a receptor and a molecular and/or cellular response (e.g., transcriptional
regulation of genes) occurs, such ligands are referred to as "agonists"
and the response produced is called "agonism." Herein,
therefore, the term agonist refers to ligands, such as estrogen,
that produce the molecular and/or cellular responses.
 Estrogens and ER play significant roles in certain human
cancers, breast cancer being one specific example. Cells in female
breast tissue normally contain ER. Interaction of estrogens with
ER in breast cells normally causes the breasts to grow at puberty
and again during pregnancy. Since breast cells normally contain
ER, it is not surprising that cells comprising tumors of the breast
also contain ER. Ninety-five percent of all breast tumors, at least
initially, have ER and are dependent on estrogens for growth. In
such breast tumor cells, estrogens acting via the ER, dramatically
escalate proliferative and metastatic activity (Osborne, et al.,
1980, Cancer, 46:2884-8.).
 Treatment of such ER-positive breast tumors comprises administration
to the individual with the tumor, compounds such as tamoxifen (TOT).
TOT can also administered to individuals who may be at high risk
for developing breast tumors in the future, for the purpose of prevention
of such tumors. Chemically, tamoxifen is one of a number of compounds
referred to as triphenyethylene derivatives. Tamoxifen is a mainstay
of breast cancer treatment and inhibits the proliferation promoting
effect of estrogens (Katzenellenbogen, et al., 1995, J Steroid Biochem
Mol Biol, 53:387-93.; Osborne, et al., 1980, Cancer, 46:2884-8.;
Jordan and Murphy, 1990, Endocr Rev, 11:578-610.). Like estrogens,
TOT binds to ER and, therefore, is also an ER ligand. Unlike estrogen
binding to ER, however, TOT binding to ER does not result in production
of significant molecular and/or cellular responses. The changes
in gene expression resulting from TOT binding to ER are significantly
less in magnitude than those resulting from estrogen binding to
ER. Such decreased responses are referred to as "partial agonism."
Ligands such as TOT, that result in partial agonism, are referred
to as "partial agonists."
 Of significance is that binding of ER by TOT prevents estrogens
from producing their effect on ER (i.e., the partial agonist precludes
effects of the agonist). Since estrogens are prevented from producing
a molecular and/or cellular response through the ER, the response
produced in the presence of both estrogens and TOT will be partial
agonism, rather than agonism. Such partial agonism is the basis
by which TOT impairs breast tumor growth (i.e., by blocking the
agonist effects of estrogens).
 With regard to TOT, while it is effective in preventing
proliferation of ER-positive breast tumor cells (i.e., cells that
contain ER) in the early stages of breast cancer treatment, such
ER-positive tumor cells invariably develop resistance to TOT. That
is, after a time (e.g., 5 years), TOT is no longer effective in
preventing estrogen stimulation of tumor proliferation and, in fact,
causes stimulation of proliferation of ER-positive tumor cells.
 The high mortality observed in breast cancer patients indicates
that additional methods and tools for diagnosing and treating breast
cancer are needed. Methods and tools for differentiating between
normal breast tissue and cells and cancerous breast tissue and cells
are desirable. Additional methods and tools for reducing or inhibiting
the growth or proliferation of breast cancer cells are also desirable.
SUMMARY OF THE INVENTION
 The present invention provides tools and methods for differentiating
normal breast tissue and cells from cancerous breast tissue and
cells. The tools are derived from a novel tumor suppressor gene
designated as Estrogen Downregulated Gene (EDG1) that is down-regulated
by estrogen in mammary epithelial cells. EDG1 encodes a protein
referred to hereinafter as the "EDG1" protein (SEQ. ID.
NO. 2). In one aspect the tool is an isolated polynucleotide which
encodes the EDG1 protein. In one embodiment, the isolated polynucleotide
comprises the nucleotide sequence of SEQ ID NO.1. The present invention
also relates to fragments of the isolated polynucleotide that can
be used as probes or primers for identifying cells that are or are
not expressing EDG1.
 In another aspect, the tool is a monoclonal antibody which
is immunospecific for the EDG1 protein. The antibody may further
comprise a detectable label, such as a fluorescent label, a chemiluminescent
label, a radiolabel or an enzyme. Also encompassed are hybridoma
cells and cell lines that produce such antibody. In another aspect,
the tool is a polyclonal sera, antibodies of which bind immunologically
to the EDG1 protein.
 In another aspect, the present invention provides a method
of detecting cancerous cells in an hormone responsive tissue test
sample. Preferably, the sample is a prostate tissue, ovarian tissue,
testes tissue, uterine tissue, cervical tissue or, more preferably
a breast tissue sample. In one embodiment, the method comprises
contacting the sample or a protein extract therefrom with at least
one antibody to the EDG1 protein under conditions wherein antibody
binding to the EDG1 protein occurs; and assaying for the presence
or absence of a complex between the antibody and a protein in the
sample, wherein a decrease in the level of the antigen-antibody
complex, as compared to the levels found in a sample of control
cells, indicates that the sample comprises cancerous cells. Preferably,
the assay is an immunocytochemical assay which permits determination
of the intracellular location of the antigen-antibody complexes.
In another embodiment, the method comprises assaying for the presence
of EDG1 transcript in the sample, wherein a decrease in the level
of the EDG1 transcript in the sample, as compared to the level of
the EDG1 transcript in a control sample, denotes that the test sample
comprises cancerous cells.
 The present invention also relates to the protein encoded
by EDG1 and biologically active or immunologically reactive fragments
thereof. In one embodiment the EDG1 protein has the amino acid sequence
of SEQ ID NO. 2. In one embodiment the EDG1 protein fragment has
the amino acid sequence of SEQ ID NO. 3.
 The present invention also provides a method for decreasing
proliferation of breast cancer cells, prostate cancer cells, testicular
cancer cells, and ovarian cancer cells. Such method comprises increasing
levels of the EDG1 protein in such cells. In one embodiment, the
cells are contacted with the EDG1 protein or a biologically active
equivalent or fragment thereof under conditions permitting uptake
of the protein or fragment. In another embodiment, the cells are
contacted with (i) a nucleic acid encoding the EDG1 protein, and
(ii) a promoter active in the cancer cell, wherein the promoter
is operably linked to the region encoding the EDG1 protein, under
conditions permitting the uptake of the nucleic acid by the cancer
cell. The cancer cell may be derived from an endocrine tissue such
as breast, ovary, prostate or testes tissue.
 The present invention also provides a method for inhibiting
the transcriptional activity of estrogen-liganded ER.alpha. in cancer
cells. Such method comprises increasing the levels of the EDG1 protein
in such cells.
BRIEF DESCRIPTION OF THE FIGURES
 FIG. 1. shows the nucleotide sequence, SEQ ID NO. 1, of
a human EDG1 cDNA and the predicted amino acid sequence, SEQ ID
NO. 2, of the EDG1 protein.
 FIG. 2. Functional interaction of EDG1 with ER.alpha.. a,
in vitro translated and [.sup.35S]methionine-labeled Estrogen Receptor
.alpha. (ER.alpha.) was incubated with GST alone or GST-EDG1 bound
to Sepharose in the presence of vehicle, 10.sup.-6 M Estradiol (E.sub.2)
or 10.sup.-6 M trans-hydroxytamoxifen (TOT). Bound protein was eluted
and analyzed by 12.5% SDS-polyacrylamide gel electrophoresis. "Input"
is an input lane and represents in vitro translated product added
in the samples. No interaction of in vitro translated products was
observed with GST alone. The autoradiograph is representative of
3 separate experiments. b, CHO cells were transfected with expression
vectors for activators (5 ng)/reporter constructs (2 .mu.g)--(ER.alpha.)/(ERE).sub.2-TATA-CAT
or Progesterone Receptor .beta. (PR .beta.)/MMTV-CAT or retinoic
acid receptor (RAR)/DR5-CAT or Gal4-VP16/G5-E1b-CAT. MCF7 cells
were transfected with (ERE).sub.2-pS2-CAT. The cells were cotransfected
with cmv5 control expression vector or increasing concentration
of an expression vector for EDG1 (cmv5-EDG1) as indicated. MCF7
cells, which expresses high endogenous ER, were transfected with
(ERE).sub.2-pS2-CAT reporter vector. CHO and MCF7 cells were also
transfected with a .beta.-galactosidase internal control reporter
to correct for transfection efficiency. Cells were then treated
for 24 h with 10.sup.-8 M estradiol (E.sub.2), 10.sup.-8 R5020,
or 10.sup.-8 all trans retinoic acid. Values are the means.+-.S.E.
from three separate experiments. c, CHO cells were transfected with
the 100 ng of pEGFP-C3-EDG1 vector or pEGFP-C3-PCMT (PCMT is a known
non-nuclear protein). For fluorescence images a fluorescein filter
was used. (Original images at 400.times.total magnification).
 FIG. 3. EDG1 expression and intracellular localization in
normal breast and breast cancer tissue and epithelial cells. a,
Total RNA was collected from untreated MCF7 cells (-), and cells
treated for 24 h with 10.sup.-9 M 17.beta.-Estradiol (E.sub.2),
10.sup.-6 M all trans retinoic acid (RA), or 5 mM hexamethylene
bisacetanide (HMBA). Total RNA was also collected from different
breast epithelial cell lines. The blot was probed with random primer-labeled
EDG1 cDNA. To control for RNA loading the same blot was reprobed
with 36B4. The autoradiographs are representative of three separate
experiments. b, Sections obtained from breast tumor and adjacent
normal breast tissue of 3 patients were stained for endogenous EDG1
using the EDG1 (peptide 152-171) polyclonal rabbit antibody and
the goat, anti-rabbit Alexa 488 secondary antibody. c, EDG1 expression
in human tissues. Master human normal blots (Invitrogen) containing
mRNA from different tissues was probed with random primer-labeled
EDG1 cDNA. To control for RNA loading the same blot was reprobed
with actin. EDG1 and .beta.-actin mRNA levels were quantified using
densitometry. EDG1 mRNA levels were normalized to .beta.-actin levels
and expressed relative to EDG1 expression in the lung.
 FIG. 4. Regulation of EDG1 intracellular localization and
relevance in breast cell growth. a, MCF10A and MCF7 cells were stained
for endogenous EDG1 b, MCF7 cells weaned out of phenol-red free-
and fall serum-containing medium for 2 weeks and treated with 10.sup.-9
M E.sub.2 or 20 ng/ml EGF for the indicated time periods. Cells
were then stained for endogenous EDG1 . c, MCF7 and MCF10A were
infected with control, EDG1, or antisense EDG1 retrovirus in the
presence or absence of tetracycline or 10.sup.-8 M Estradiol (E.sub.2).
Five days after infection, cells were stained for EDG1 expression
and cell number was determined using the CellTiter 96 Aqueous One
Solution Proliferation Assay. Values for cell number are expressed
relative to the absorbance in control cells grown in the presence
of tetracycline (which is set at 1). Values are the means.+-.S.E.
from two separate experiments with triplicate wells for each group.
d, MCF7 cells were infected with control, EDG1 or EDG1 .sub.AS retrovirus
in the presence of 3 .mu.g/ml tetracycline. Twenty-four hours later
cells were given fresh media tetracycline. Four days later cells
were detached and plated for anchorage independent growth. Values
are expressed as the number of colonies formed per number of cells
plated.times.100. Values are expressed relative to the number of
colonies/cells plated for cells infected with control retroviruses
grown in the presence of tetracycline (which is set at 1). Values
are the means.+-.S.E. from two separate experiments with triplicate
wells for each group. e, MCF10A and MDA-MD-231 cells were infected
with retroviruses and plated for proliferation or immunostaining
as described in (d). In a, b, c, and e, cells were stained for endogenous
EDG1 using the EDG1 (peptide 152-171) polyclonal rabbit antibody
and the goat, anti-rabbit Alexa 488 secondary antibody. Cells were
viewed under a fluorescence microscope at 200.times.magnification.
MCF10A cells were also stained with nile red to examine lipid vacoule
 FIG. 5. Functional interaction of EDG1 with binding proteins
for components of the extracellular matrix and regulation of anchorage
independent growth. a, in vitro translated and [.sup.35S]methionine-label-
ed Integrin .beta.4 Receptor or Laminin Binding Protein were incubated
with GST alone or GST-EDG1 bound to Sepharose. Bound protein was
eluted and analyzed by 12.5% SDS-polyacrylamide gel electrophoresis.
The numbers at the right indicate molecular size markers in kilodaltons.
"Input" is an input lane and represent 10% of total in
vitro translated products added in the samples. The autoradiograph
is representative of 3 separate experiments. b, MCF7 infected with
control, EDG1 or EDG1.sub.As retroviruses were immunostained using
67LR IgG monoclonal mouse antibody and goat, anti-mouse Alexa 594
secondary antibody and EDG1 polyclonal rabbit IgG antibody and goat,
anti-rabbit Alexa 488 secondary antibody. c, MCF7 cells were transfected
with 100 ng of pEGFP-EDG1 , pRFP-67LR, pEGFP-C3, pRFP-C1, or pEGFP-hPMC2
as indicated. pRFP-C1 and pEGFP-C3 are control fluorescent protein
vectors without a cDNA insert and pEGFP-hPMC2 is an unrelated nuclear
protein. The cells were observed under the microscope 24 h later.
For fluorescence images rhodamine or fluorescein filters were used.
Original images are at 400.times.total magnification.
DETAILED DESCRIPTION OF THE INVENTION
 As used herein the following terms have the following meanings:
 "Antibody" means a protein molecule that binds
to, cross reacts with, or is immunoreactive with a specific antigen
or immunogen. The binding reaction between an antibody and its antigen
is specific in that the antibody binds only to an amino acid sequence
present within the specific protein (i.e., an epitope). An anti-EDG1
antibody means an antibody molecule that binds to one or more epitopes
of the EDG1 protein.
 "Biological sample" means a sample of mammalian
cells. These cells may be part of a tissue or organ sample obtained,
for example, by biopsy, or they may be individual cells, for example,
blood cells or cells grown in tissue culture.
 "Cancer cell" or "cancerous cell" means
a cell in or from a carcinoma.
 "Breast cancer" means any of various carcinomas
of the breast or mammary tissue.
 "cDNA" means a DNA prepared using messenger RNA
(mRNA) as template. The advantage of using a cDNA, as opposed to
genomic DNA or DNA polymerized from a genomic, non- or partially-processed
RNA template, is that the cDNA primarily contains coding sequences
of the corresponding protein.
 "Expression" means the production of a protein
or a gene transcript (i.e. mRNA) in a cell.
 "Hormone responsive tissue" as used herein refers
to tissues that are normally responsive to estrogens or androgens.
Hormone responsive tissues include the mammary glands, testes, prostate,
uterus and cervix. A tissue which is normally responsive to estrogens
or androgens may lose its responsiveness to the hormone. Thus, "hormone
responsive tissue" is a broad term as used herein and encompasses
both hormone-sensitive and hormone insensitive tissues.
 "Estrogen-receptor positive" as used herein refers
to a cell that comprises estrogen receptor and is responsive to
estrogen and to agents that bind to the estrogen receptor, such
 "Estrogen-receptor negative" as used herein refers
to a cell that is normally responsive to estrogen, such as a mammary
epithelial cell, but that contains little to no estrogen receptor.
As a result, the estrogen receptor negative cell is estrogen-insensitive
and refractory to treatment with tamoxifen.
 "Label" means to incorporate into a compound a
substance that is readily detected. Such substances include radioactive
substances and fluorescent dyes, for example.
 "Native" means the nucleic acid of a non-mutated
gene or peptide sequence encoded by such a gene as found in a phenotypically
 "Neoplasia" means the process resulting in the
formation and growth of an abnormal tissue that grows by cellular
proliferation more rapidly than normal, and continues to grow after
the stimuli that initiated the new growth ceases.
 "Normal cell" means a non-cancerous cell.
 "Proliferation" means growth and reproduction,
i.e., division of cells
 "Tumor" refers to a spontaneous, new growth of
tissue in the body that forms an abnormal mass. Tumors are comprised
of cells and such cells are known as tumor cells. Tumors and cells
derived from tumors can be either benign or malignant. Cells that
are malignant have a variety of properties that benign cells and
non-tumor cells do not have. Malignant cells invade, grow and destroy
adjacent tissue, metastasize, and usually grow more rapidly than
benign tumor cells. "Neoplasm" is essentially synonymous
 "Tumor suppressor gene" refers to a gene whose
expression within a tumor cell suppresses the ability of such cells
to grow spontaneously and form an abnormal mass.
 All publications and other references mentioned herein are
incorporated by reference in their entirety.
 EDG1 Protein
 The present invention provides a protein referred to hereinafter
as EDG1 protein and functional equivalents thereof The EDG1 protein
is encoded by the tumor suppressor gene designated Estrogen Downregulated
 Intracellular Localization of EDG1 Protein
 EDG1 is localized in different intracellular compartments
in normal breast and breast cancer epithelial cells. (See FIG. 4)
In normal mammary epithelial cells, represented by the cell line
MCF10A, EDG1 is localized primarily in the nucleus. In contrast,
lower and more diffuse cytoplasmic staining occurs in MCF7 cells,
which are representative of estrogen receptor positive breast cancer
epithelial cell (FIG. 4A). Interestingly, EDG1 localized primarily
to the nucleus when MCF7 cells were weaned out of their maintenance
medium containing phenol red and full serum to phenol red-free media
containing charcoal-stripped serum (FIG. 4B). Estradiol, (E.sub.2)
and epidermal growth factor (EGF) affect the levels and/or intracellular
localization of EDG1 protein in breast cancer cells. A slight decrease
in EDG1 expression is evident 12 h and 16 h after E.sub.2 treatment.
(FIG. 4B) After treatment with either Epidermal Growth Factor (EGF)
or E.sub.2, a decrease in the nuclear localization of EDG1 was evident
in MCF7 cells (FIG. 4B). Estrogen- and EGF-induced nuclear export
of EDG1 is inhibited by antiestrogen ICI182,780 and Mitogen-Activated
Protein Kinase Kinase (MAPKK) inhibitor PD098,059 respectively (data
not shown). It was observed that EGF, not E.sub.2, induces EDG1
nuclear export in MCF10A cells which expresses very low levels of
estrogen receptor (ER) (data not shown).
 Interaction of EDG1 Protein with Other Cellular Proteins
 EDG1 interacting proteins were identified using the yeast
two-hybrid system and the interactions were verified in vitro using
GST pull-down assays (FIG. 5A). The strongest interactors from the
yeast two hybrid screenings are two proteins involved in cell adhesion--the
67 kD laminin receptor (67LR) and the integrin .beta.4 interactor
protein (p27/BBP). Stronger interaction of EDG1 with p27/BBP, when
compared to its interaction with 67LR, was observed in GST-pull
down assays. Both 67LR and p27/BBP have been proposed to be part
of the structural link between extracellular matrix proteins and
the cytoskeleton (Biffo S, Sanvito F, Costa S, Preve L, Pignatelli
R, Spinardi L, Marchisio PC (1997) Isolation of a novel beta4 integrin-binding
protein (p27(BBP)) highly expressed in epithelial cells. J. Biol.
Chem. 272: 30314-30321; Ardini E, Tagliabue E, Magnifico A, Buto
S, Castronovo V, Colnaghi MI, Menard S. (1997) Co-regulation and
physical association of the 67-kDa monomeric laminin receptor and
the alpha6beta4 integrin. J. Biol. Chem. 272: 2342-2345). The functional
relatedness of these two proteins supports the biological relevance
of the interaction of EDG1 with these proteins.
 To determine the functional consequence of the interaction
of EDG1 with 67LR, the expression of 67LR was examined in cells
infected with control or EDG1 retroviruses. 67LR is a cell surface-associated
protein that interacts specifically and directly with laminin. Increased
cell surface expression of 67LR is associated with increased invasiveness
and less differentiated phenotypes of several types of human malignancies
(Sanvito F, Vivoli F, Gambini S, Santambrogio G, Catena M, Viale
E, Veglia F, Donadini A, Biffo S, Marchisio pc. (2000) Expression
of a highly conserved protein, p27bbp, during the progression of
human colorectal cancer. Cancer Res. 60: 510-516; Cress AE, Rabinovitz
I, Zhu W, Nagle RB. (1995) The alpha 6 beta 1 and alpha 6 beta 4
integrins in human prostate cancer progression. Cancer Metastasis
Rev. 14: 219-228.). Increased cell surface expression of 67LR is
seen in breast cancer cells treated with estrogen and progesterone
(Castronovo V, Taraboletts G, Liotta LA, Sobel M (1989) Modulation
of Laminin receptor Expression by Estrogen and Progestins in Human
Breast Cancer Cell Lines, J. Nat. Cancer Instit. 81: 781-788.).
The role of 67LR in breast cancer is not limited to invasion because
breast cancer cells undergoing proliferation express increased cell
surface 67LR. While 67LR shows primary membrane localization in
control cells, cytoplasmic staining was also evident (FIG. 5B).
Cells infected with EDG1 retroviruses show an overall decrease in
67LR expression, especially in the membrane. Cells that were treated
with estradiol, which induces EDG1 nuclear export, show increased
67LR staining. 67LR has been proposed to originate from a 37 kDa
precursor ribosomal protein (37LRP) that can be localized to the
nucleus and the cytoplasm (Ardini E, Posole G, Tagliabue E, Magnifico
A, Castronovo V, Sobel ME, Colnaghi MI, Menard S. (1998) the 67-kDa
laminin receptor originated from a ribosomal protein that acquired
a dual function during evloution, Mol. Biol. Evol. 15:1017-1025).
Acylation of 37LRP leads to the formation of the 67LR dimer and
the acquisition of laminin binding capacity. Thus it is possible
that EDG1 interferes with 67LR processing. Because the antibody
utilized in these experiments does not detect 37LRP, an expression
vector wherein 67LR cDNA was cloned in frame with Red Fluorescent
Protein (RFP) (pRFP-67LR) was used to explore this possibility.
In cells transfected with pRFP-67LR, red fluorescence can be localized
throughout the cell (FIG. 4C). Interestingly, when RFP-67LR was
coexpressed with green fluorescence protein-tagged EDG1 the localization
of red fluorescence was more limited, showing localization primarily
in the nucleus. The change in localization was specific to cells
expresssing EDG1. No change in red fluorescence localization was
evident in cells cotransfected with control GFP expression vector
(i.e. no EDG1 cDNA) or with an unrelated protein that we have previously
reported to be primarily nuclear (GFP-hPMC2, Montano MM, Wittman,
BM, Bianco NR (2000) Identification and Characterization of a Novel
Factor that Regulates Quinone Reductase Gene Transcriptional Activity,
J. Biol. Chem. 275: 34306-34313). The data support an effect of
EDG1 on 67LR processing.
 Structure of EDG1 Protein
 In one embodiment, the EDG1 protein is 359 amino acids in
length and comprises the amino acid sequence, SEQ ID NO. 2, shown
in FIG. 1. The EDG1 protein has a nuclear localization signal spanning
residues 150-177. DNAStar analyses predict that the EDG1 protein
consists mostly of alpha helices interspersed with turns and coils.
EDG1 protein is a highly hydrophilic and highly charged, with a
large proportion of the amino acids surface exposed. Many of the
alpha helices are amphipathic (i.e. negatively or positively charged).
Positive charges come from runs of triple arginine or lysines and
negative charges come from triple runs of aspartate or glutamate.
These indicate the potential for electrostatic interactions coming
about from protein-protein or protein-nucleic acid contacts.
 EDG1 Protein Functional Equivalents
 The present invention also encompasses functional equivalents
of the EDG1 protein that may vary structurally from the EDG1 protein
(SEQ. ID. NO. 2), but have equivalent function. Such functional
equivalents are immunologically cross reactive or biologically active
equivalents of the EDG1 protein which comprises SEQ ID NO. 2. Such
functional equivalents have an altered sequence in which one or
more of the amino acids in the corresponding reference sequence
is substituted or in which one or more amino acids are deleted from
or added to the corresponding reference sequence.
 While it is possible to have nonconservative amino acid
substitutions, it is preferred that, except for the substitutions
that are made to replace cysteine, the substitutions be conservative
amino acid substitutions, in which the substituted amino acid has
similar structural or chemical properties with the corresponding
amino acid in the reference sequence. By way of example, conservative
amino acid substitutions involve substitution of one aliphatic or
hydrophobic amino acids, e.g., alanine, valine, leucine and isoleucine,
with another; substitution of one hydroxyl-containing amino acid,
e.g., serine and threonine, with another; substitution of one acidic
residue, e.g., glutamic acid or aspartic acid, with another; replacement
of one amide-containing residue, e.g., asparagine and glutamine,
with another; replacement of one aromatic residue, e.g., phenylalanine
and tyrosine, with another; replacement of one basic residue, e.g.,
lysine, arginine and histidine, with another; and replacement of
one small amino acid, e.g., alanine, serine, threonine, methionine,
and glycine, with another.
 Preferably, the deletions and additions are located at the
amino terminus, the carboxy terminus, or both, of SEQ ID NO. 2.
As a result of the alterations, the EDG1 functional equivalent has
an amino acid sequence which is at least 90% identical, preferably
at least 95% identical, more preferably at least 97% identical to
SEQ ID NO. 2 Sequences which are at least 90% identical have no
more than 1 alteration, i.e., any combination of deletions, additions
or substitutions, per 10 amino acids of the reference sequence.
Percent identity is determined by comparing the amino acid sequence
of the variant with the reference sequence using MEGALIGN project
in the DNA STAR program.
 In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte & Doolittle, (1982) A
simple method for displaying the hydropathic character of a protein.
J. Mol. Biol. 157:105-132). It is accepted that the relative hydropathic
character of the amino acid contributes to the secondary structure
of the resultant protein, which in turn defines the interaction
of the protein with other molecules, for example, enzymes, substrates,
receptors, DNA, antibodies, antigens, and the like.
 It is known in the art that certain amino acids may be substituted
by other amino acids having a similar hydropathic index or score
and still result in a protein with similar biological activity,
i.e., still obtain a biological functionally equivalent protein.
It is also understood in the art that the substitution of like amino
acids can be made effectively on the basis of hydrophilicity. U.S.
Pat. No. 4,554,101, incorporated herein by reference, states that
the greatest local average hydrophilicity of a protein, as governed
by the hydrophilicity of its adjacent amino acids, correlates with
a biological property of the protein.
 It is understood that an amino acid can be substituted for
another having a similar hydrophilicity value and still obtain a
biologically active and immunologically cross-reactive protein.
As outlined above, amino acid substitutions are generally based
on the relative similarity of the amino acid side-chain substituents,
for example, their hydrophobicity, hydrophilicity, charge, size,
and the like.
 The immunologically cross-reactive EDG1 variants immunologically
bind to one or more of the antibodies that are raised using the
EDG1 protein as an immunogen. The biologically active EDG1 variants
inhibit or reduce estrogen-bound estrogen receptor transcriptional
activity in MCF7 cells and proliferation of normal mammary epithelial
cells or cancerous mammary epithelial cells.
 While it is difficult to predict the exact effect of the
substitution, deletion or insertion in advance of doing so, for
example, when modifying an immune epitope on the EDG1 protein, one
skilled in the art will appreciate that the effect will be evaluated
by routine screening assays. For example, a change in the immunological
character of the EDG1 protein, such as affinity for a given antibody,
is measured by a competitive-type immunoassay. Modifications of
protein properties such as redox or thermal stability, hydrophobicity,
susceptibility to proteolytic degradation, or the tendency to aggregate
with carriers or into multimers may be assayed by methods well known
to one of skill in the art.
 The present invention also encompasses fusion proteins comprising
the EDG1 protein or a functional equivalent thereof and a tag, i.e.,
a second protein or one or more amino acids, preferably from about
2 to 65 amino acids, more preferably from about 34 to about 62 amino
acids, which are added to the amino terminus of, the carboxy terminus
of, or any point within the amino acid sequence of the EDG1 protein,
or a variant of such protein. Typically, such additions are made
to stabilize the resulting fusion protein or to simplify purification
of an expressed recombinant form of the corresponding EDG1 protein
or variant of such protein. Such tags are known in the art. Representative
examples of such tags include sequences which encode a series of
histidine residues, the epitope tag FLAG, the Herpes simplex glycoprotein
D, beta-galactosidase, maltose binding protein, or glutathione S-transferase.
 The EDG1 protein and functional equivalents thereof may
be produced by conventional peptide synthesizers. The EDG1 proteins
and functional equivalents thereof may also be produced using cell-free
translation systems and RNA molecules derived from DNA constructs
that encode the EDG1 proteins. Alternatively, EDG1 proteins and
functional equivalents thereof are made by transfecting host cells
with expression vectors that comprise a DNA sequence that encodes
the respective EDG1 protein or functional equivalents and then inducing
expression of the protein in the host cells. For recombinant production,
recombinant constructs comprising a sequence which encodes the EDG1
protein or functional equivalents thereof are introduced into host
cells by conventional methods such as calcium phosphate transfection,
DEAE-dextran mediated transfection, transvection, microinjection,
cationic lipid-mediated transfection, electroporation, transduction,
scrape loading, ballistic introduction or infection.
 The EDG1 protein and functional equivalents thereof may
be expressed in suitable host cells, such as for example, mammalian
cells, yeast, bacteria, or other cells under the control of appropriate
promoters using conventional techniques. Following transformation
of the suitable host strain and growth of the host strain to an
appropriate cell density, the cells are harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification of the EDG1 protein.
 Conventional procedures for isolating recombinant proteins
from transformed host cells, such as isolation by initial extraction
from cell pellets or from cell culture medium, followed by salting-out,
and one or more chromatography steps, including aqueous ion exchange
chromatography, size exclusion chromatography steps, and high performance
liquid chromatography (HPLC), and affinity chromatography may be
used to isolate recombinant EDG1 protein.
 EDG1 Polypeptides and Oligopeptides
 The present invention also encompasses oligopeptides or
polypeptides, referred to hereinafter collectively as "EDG1
polypeptides," that are less than 359 amino acids in length
and comprise a consecutive sequence in SEQ ID NO. 2. In one aspect,
the EDG1 polypeptides are immunologically cross-reactive with the
EDG1 protein. Such polypeptides can be used to prepare antibodies
that form antigen-antibody complexes with the EDG1 protein. In one
embodiment, the EDG1 polypeptide comprises amino acids 152-171 of
SEQ ID NO. 2. In other words, the EDG1 polypeptide comprises the
hydrophilic region, KHRRRPSKKKRHWKPYYKL SEQ ID NO. 3.
 In another aspect, the EDG1 polypeptide has the biological
activity of the native EDG1 protein, i.e., the EDG1 polypeptide
has the ability to reduce or inhibit proliferation of a non-cancerous
or cancerous mammary epithelial cell.
 The present invention provides isolated polynucleotides
which encode the EDG1 protein or a functional equivalent thereof.
The EDG1 polynucleotides may be single-stranded or double stranded.
Such polynucleotides may be DNA or RNA molecules In one embodiment
the isolated polynucleotide comprises the EDG1 cDNA sequence, SEQ
ID NO. 1, shown in FIG. 1. Sequence analysis of the EDG1 cDNA clone
indicates an open reading frame of 1077 bp (359 amino acids) encoding
a 40 kDa protein. The EDG1 polynucleotides are useful for preparing
 In vivo, EDG1 acts as a tumor suppressor gene. Database
searches indicate that EDG1 can be localized to chromosome arm 17q.
EDG1 mRNA expression is prevalent in normal mammary epithelial cells
and in other human hormone responsive tissues such as the ovary
and prostate, and testes (FIG. 2C). Expression of EDG1 mRNA is low
in breast cancer epithelial cells. Estradiol or E.sub.2 which induces
breast cancer cell growth, has an inhibitory effect on EDG1 mRNA
expression in breast cancer cells. Conversely, hexamethylene-bis-acetamide
(HMBA), which is known to be an inducer of differentiation and apoptosis,
upregulates EDG1 mRNA expression in breast cancer cells (FIG. 3A).
 The present invention also encompasses isolated polynucleotides
whose sequence is the complement of the EDG1 cDNA sequence, SEQ
ID NO. 1, and polynucleotides that hybridize under stringent conditions,
preferably under highly stringent conditions, to the open reading
frame sequence of the EDG1 cDNA sequence, SEQ ID NO. 1, or the complement
thereof. Such hybridization conditions are based on the melting
temperature, Tm, of the nucleic acid binding complex or probe, as
described in Berger and Kimmel (1987) Guide to Molecular Cloning
Techniques, Methods in Enzymology, vol 152, Academic Press. The
term "stringent conditions," as used herein, is the "stringency"
which occurs within a range from about Tm-5 (5.degree. below the
melting temperature of the probe) to about 20.degree. C. below Tm.
As used herein, "highly stringent" conditions employ at
least 0.2.times.SSC buffer and at least 65.degree. C. As recognized
in the art, stringency conditions can be attained by varying a number
of factors such as the length and nature, i.e., DNA or RNA, of the
probe; the length and nature of the target sequence, the concentration
of the salts and other components, such as formamide, dextran sulfate,
and polyethylene glycol, of the hybridization solution. All of these
factors may be varied to generate conditions of stringency which
are equivalent to the conditions listed above.
 Variations in the above conditions may be accomplished through
the inclusion and/or substitution of alternate blocking reagents
used to suppress background in hybridization experiments. Typical
blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured
salmon sperm DNA, and commercially available proprietary formulations.
The inclusion of specific blocking reagents may require modification
of the hybridization conditions described above, due to problems
 The present invention also relates to polynucleotide encoding
a protein having a sequence that is at least 85%, preferably at
least 90%, more preferably at least 95%, most preferably at least
97% identical to the amino acid sequences depicted in FIG. 1 and
set forth in SEQ ID NO. 2, provided that such protein is an immunologically
cross-reactive or biologically reactive equivalent of the EDG1 protein.
Such sequences include allelic variants, species variants and other
amino acid sequence variants (e.g., including "muteins"
or "mutant proteins"), whether naturally-occurring or
 Polynucleotides that encode the EDG1 protein and sequences
which are the complements thereof are useful tools for designing
hybridization probes for screening tissue samples, particularly
tissues from patients known to have or suspected of having breast
cancer, and for isolating and identifying cDNA clones and genomic
clones encoding the EDG1 genes or allelic forms thereof. Such hybridization
techniques are known to those of skill in the art. SEQ ID NO. 1,
and sequences which are the complement thereof are also useful for
designing primers for polymerase chain reaction (PCR), a technique
useful for obtaining large quantities of cDNA molecules that encode
the EDG1 proteins.
 Also encompassed by the present invention, are single stranded
polynucleotides, hereinafter referred to as antisense polynucleotides,
having sequences which are complementary to the DNA and RNA sequences
which encode the EDG1 protein. The term complementary as used herein
refers to the natural binding of the polynucleotides, through hydrogen
bond formation between complementary nucleotide bases, under permissive
salt and temperature conditions by base pairing.
 The present invention also provides primers which can be
used in PCR to obtain the EDG1 poylnucleotides from cDNA libraries,
for screening tissue samples, or for diagnostic purposes. The present
invention also encompasses oligonucleotides that are used as primers
in polymerase chain reaction (PCR) technologies, reverse transcriptase-PCR
(RT-PCR) for example, to amplify transcripts of the genes which
encode the EDG1 proteins or portions of such transcripts. Preferably,
the primers comprise 12-50 nucleotides, more preferably 15-30 nucleotides.
Preferably, the primers have a G+C content of 40% or greater. Such
oligonucleotides are at least 98% complementary with a portion of
the DNA strand, i.e., the sense strand, which encodes the respective
EDG1 or a portion of its corresponding antisense strand. Preferably,
the primer has at least 99% complementarity, more preferably 100%
complementarity, with such sense strand or its corresponding antisense
strand. Primers which have 100% complementarity with the antisense
strand of a double-stranded DNA molecule which encodes a EDG1 protein
have a sequence which is identical to a sequence contained within
the sense strand. The identity of primers which are 15 nucleotides
in length and have full complementarity with a portion of the antisense
strand of a double-stranded DNA molecule which encodes the EDG1
protein and is determined using the nucleotide sequence, SEQ ID
NO: 1, shown in FIG. 1.
 Such primers for PCR comprise a pair of set of primers.
One primer of the pair is called the "forward primer"
and is located at the left end of the sequence to be amplified.
The second primer of is called the "reverse primer" and
is located at the right end of the sequence to be amplified. The
forward primer hybridizes to the opposite strand of the template
(the DNA to be amplified) than does the reverse primers. Selection
of forward and reverse primers, for the purpose of amplifying a
sequence of DNA by PCR, is well known to one skilled in the art.
 The present invention also encompasses oligonucleotides
that are useful as hybridization probes for detecting transcripts
of the genes which encode the EDG1 protein Preferably, such oligonucleotides
comprise at least 200 nucleotides. Such hybridization probes have
a sequence which is at least 90% complementary with a contiguous
sequence contained within the sense strand or antisense strand of
a double stranded DNA molecule which encodes the EDG1 protein. Such
hybridization probes bind to the sense strand or antisense under
stringent conditions, preferably under highly stringent conditions.
The probes are used in Northern assays to detect transcripts of
EDG1 homologous genes and in Southern assays to detect EDG1 homologous
genes. The identity of probes which are 200 nucleotides in length
and have full complementarity with a portion of the sense or antisense
strand of a double-stranded DNA molecule which encodes the EDG1
protein is determined using the nucleotide sequence, SEQ ID NO:1,
shown in FIG. 1.
 The present invention also encompasses isolated polynucleotides
which are alleles of the genes which encode the EDG1 proteins. As
used herein, an allele or allelic sequence is an alternative form
of the gene which may result from one or more mutations in the sequences
which encode the EDG1 proteins. Such mutations typically arise from
natural addition, deletion of substitution of nucleotides in the
open reading frame sequences Any gene may have none, one, or several
allelic forms. Such alleles are identified using conventional techniques,
such as for example screening libraries with probes having sequences
identical to or complementary with one or more EDG1 polynucleotides.
 The present invention also encompasses altered polynucleotides
which encode the EDG1 protein or a functional equivalent of the
EDG1 protein. Such alterations include deletions, additions, or
substitutions. Such alterations may produce silent changes and result
in an EDG1 protein having the same amino acid sequence as the EDG1
protein encoded by the unaltered polynucleotide. Such alterations
may produce a nucleotide sequence possessing non-naturally occurring
codons. For example, codons preferred by a particular prokaryotic
or eucaryotic host may be incorporated into the nucleotide sequences
shown in FIG. 1 to increase the rate of expression of the proteins
encoded by such sequences. Such alterations may also introduce new
restriction sites into the sequence or result in the production
of a EDG1 protein variant. Typically, such alterations are accomplished
using site-directed mutagenesis.
 Synthesis of Polynucleotides Encoding EDG1 Proteins or Variants
 Polynucleotides comprising sequences encoding a EDG1 protein
or a functional equivalent thereof may be synthesized in whole or
in part using chemical methods. Polynucleotides which encode an
EDG1 protein, particularly alleles of the genes which encode an
EDG1 protein, may be obtained by screening a genomic library or
cDNA library with a probe comprising sequences identical or complementary
to the sequences shown in FIG. 1 or with antibodies immunospecific
for an EDG1 protein to identify clones containing such polynucleotide.
 The probes are used in Northern blot or colony hybridization
assays under high stringency conditions. Alternatively, polynucleotides
encoding EDG1 proteins may be made using polymerase chain reaction
(PCR) technology and primers which bind specifically to sequences
which are known to encode a EDG1 protein.
 The present invention also provides antibodies that are
immunospecific for the EDG1 protein. As used herein the term immunospecific
means the antibody binds with greater affinity to an EDG1 protein
than other proteins that are found in normal breast cells.
 The term "antibody" encompasses monoclonal antibodies,
polyclonal antibodies, multispecific antibodies (e.g., bispecific
antibodies), and antibody fragments, so long as they exhibit the
desired biological activity. "Antibody fragments" comprise
a portion of a full length antibody, generally the antigen binding
or variable region thereof. Examples of antibody fragments include
Fab, Fab', F(ab').sub.2, and Fv fragments.
 Antibodies raised against EDG1 are produced by immunizing
a host animal with an EDG1 protein or an antigenic fragment thereof.
Suitable host animals for injection of the protein immunogen include,
but are not limited to, rabbits, mice, rats, goats, and guinea pigs.
Various adjuvants may be used to increase the immunological response
in the host animal. The adjuvant used depends, at least in part,
on the host species. For example, guinea pig albumin is commonly
used as a carrier for immunizations in guinea pigs. Such animals
produce heterogenous populations of antibody molecules, which are
referred to as polyclonal antibodies and which may be derived from
the sera of the immunized animals.
 The term "monoclonal antibody" as used herein
refers to an antibody obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible naturally-occurring
mutations that may be present in minor amounts. Monoclonal antibodies
are highly specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional (polyclonal) antibody preparations,
which typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed against
a single determinant on the antigen.
 The monoclonal antibodies to be used in accordance with
the present invention may be made by the hybridoma method, first
described by Kohler et al., Nature 256: 495 (1975), in which case
the hybridoma cell lines that are obtained secrete the monoclonal
antibodies during growth. In order to grow the hybridoma cell lines
and obtain the secreted antibodies, the hybridoma cell lines may
be grown in cell culture and culture medium containing the monoclonal
antibodies collected. Alternatively, the hybridoma cell lines may
be injected into, and grown within, the peritoneal cavity of live
animals, preferably mice. As the hybridoma cell lines grow within
the peritoneal cavity of the animal, the monoclonal antibodies are
secreted. This peritoneal fluid, called "ascites," is
collected using a syringe to obtain the monoclonal antibodies. Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, Iga, IgD and any class thereof.
 Antibody preparations may be isolated or purified. An "isolated"
antibody is one which has been identified and separated and/or recovered
from a component of its natural environment. Contaminant components
of its natural environment are materials which would interfere with
diagnostic or therapeutic uses for the antibody, and may include
enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
In preferred embodiments, the antibody may be purified (1) to greater
than 95% by weight of antibody as determined by the Lowry method,
and most preferably more than 99% by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using Coomassie
blue or, preferably, silver stain. Isolated antibody includes the
antibody in situ within recombinant cells since at least one component
of the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one purification
 Antibodies immunospecific for EDG1 are useful diagnostic
markers detecting cancerous epithelial cells in a tissue selected
from breast tissue, ovarian tissue, testicular tissue, prostate
tissue, uterine tissue and cervical tissue. In accordance with the
present invention, it has been shown that cancerous mammary epithelial
cells have lower levels of EDG1 protein than non-cancerous mammary
epithelial cells. The diagnostic method comprises the steps of contacting
a sample of test cells or a protein extract thereof with immunospecific
anti-EDG1 antibodies and assaying for the formation of a complex
between the antibodies and a protein in the sample. Because EDG1
protein localizes to the nucleus in non-cancerous mammary epithelial
cells and, if present, to the cytoplasm of cancerous mammary epithelial
cells, it is preferred that the assay be an immunocytochemical assay.
The cells may be fixed or premeablized to permit interaction between
the antibody and intracellular proteins. Interactions between antibodies
and a protein or peptide in the sample are detected by radiometric,
colorimetric, or fluorometric means. Detection of the antigen-antibody
complex may be accomplished by addition of a secondary antibody
that is coupled to a detectable tag, such as for example, an enzyme,
fluorophore, or chromophore. Formation of low levels of complex
in the test cell as compared to the normal cells indicates that
the test cell is cancerous.
 Cancer Detection Methods Employing EDG1 Polynucleotides
 The EDG1 polynucleotides or fragments are also useful for
detecting, defining the borders of, or grading mammary epithelial
cell carcinomas in patients known to have or suspected of having
a mammary epithelial cell carcinoma. The EDG1 polynucleotides or
fragments may also be used to detect cancerous cells in prostate,
testicular, and ovarian tissue. In accordance with the present invention,
it has been determined that mammary epithelial cell lines derived
from mammalian tissues obtained from individuals with breast cancer
have lower levels of EDG1 mRNA than mammary epithelial cells derived
from normal mammary tissues. In accordance with the present invention,
it has also been determined that cells derived from prostate tissue,
testicular tissue, and ovarian tissue contain relatively high levels
of EDG1 transcript.
 Thus, the polynucleotides of the present invention may be
used as probes in Northern analysis to identify tissues which have
comparatively lower and higher levels of EDG1 mRNA. In such procedures
total RNA or mRNA is obtained from the cells that are know to be
or suspected of being cancerous and from non-cancerous cells, i.e.
breast epithelial cells, testicular epithelial cells, prostate cells,
or ovarian cells, preferably from the same patient, and then assayed
using the EDG1-designed probe. In general, the non-cancerous cells
will be obtained from tissues near but outside the border of the
 In one example, the coding sequence is radioactively labeled
with .sup.32P or digoxigenin, and then hybridized in solution to
RNA that is isolated from test cells, e.g., mammary epithelial cells
suspected of being cancerous, and separated by size using gel electrophoresis
and blotted to nitrocellulose paper. After hybridization and washing
of the nitrocellulose paper, hybridization of the EDG1 probe to
RNA on the nitrocellulose, as revealed by autoradiography, indicates
expression of the EDG1 mRNA. Decreased levels of EDG1 mRNA expression
in the test cells as compared to levels of EDG1 mRNA present in
normal epithelial cells derived from the same type of tissue indicates
that the test cells are cancerous.
 In another embodiment of the present invention, EDG1 probes,
labeled as described above, are used to hybridize directly to test
cells, e.g. mammary epithelial cells or tissues suspected of being
cancerous, and to normal cells derived from the same type of tissue,
i.e. control cells. The cells or tissues are fixed before hybridization,
using procedures well known to those skilled in the art. Hybridization
is performed under conditions similar to those described above.
Detection of hybridization, by autoradiography for example, indicates
the presence of EDG1 transcripts within the cells or tissues. A
reduced level of EDG1 transcripts in the test tissues or cells as
compared to control cells indicates that the test cells are cancerous.
 Similarly, EDG1-designed primers may be used in RT-PCR to
quantify the amount of EDG1 mRNA in the test tissues and cells.
Examples of such primers include, but are not limited to (for EDG1)
1 rt1: cagtgtgatttctagagc, SEQ ID NO. 4, and rt2: agagcagaactactcaag,
SEQ ID NO. 5.
 Alternatively, EDG1-designed primers may be used to analyze
tissue sections from human patients by an RT in situ-PCR hybridization
protocol as described Nuovo et al (1994) in Am J. Pathol., 144,
659-666, which is specifically incorporated herein by reference.
 Cancer Detection Methods Employing Anti-EDG1 Antibodies
 Anti-EDG1 antibodies have a diagnostic use, since simple
immunochemical staining of tissue sections, cells, and protein extracts
derived from mammary, prostate, testicular, and ovarian tissues
can be used to estimate the portion of cells expressing the EDG1
protein. Such a test based on the use of anti-EDG1 antibodies and
other standard secondary techniques of visualization will be useful
in cancer diagnosis, particularly cancer diagnosis of breast tissue.
Such a test of tumor suppressor gene expression might also be useful
to the scientific research community.
 In a diagnostic method of the present invention, the anti-EDG1
antibodies are used to determine the extent to which EDG1 protein
is present in a tissue sample obtained from an individual known
to have or suspected of having carcinoma, particularly breast carcinoma.
This can be determined using known techniques. Comparison of results
obtained from the tissue sample with results obtained from an appropriate
control (e.g., cells or tissue of the same type known to have normal
EDG1 levels) is carried out. Decreased EDG1 levels are indicative
of an increased probability of abnormal cell proliferation or oncogenesis
or of the actual occurrence of abnormal proliferation or oncogenesis.
It is contemplated that the levels of EDG1 in cancerous cells will
be at least 50% less than the level of EDG1 protein in non-cancerous
cells, more preferably the levels will be less than 30% of normal
levels, most preferably EDG1 will not be expressed. In accordance
with the present invention, it has been shown that cells derived
from more advanced carcinomas will have lower levels of EDG1 than
cells derived from less advanced carcinomas. Thus, the levels of
EDG1 in the test cells can be used as a prognostic marker of the
 The sample may be untreated, or subjected to precipitation;
fractionation, separation, or purification before combining with
the anti-EDG1 protein antibody. In those cases where proteins are
extracted from the sample, it is preferred that isolated proteins
from the sample be attached to a substrate such as a column, plastic
dish, matrix, or membrane, preferably nitrocellulose. For isolated
protein, the preferred detection method employs an enzyme-linked
immunosorbent assay (ELISA) or a Western immunoblot procedure.
 Formation of the complex is indicative of the presence of
the EDG1 protein in the test sample. Thus, the method is used to
determine whether there is a decrease or increase in the levels
of the EDG1 protein in a test sample as compared to levels of the
EDG1 protein in a control sample and to quantify the amount of the
EDG1 protein in the test sample. Deviation between control and test
values establishes the parameters for diagnosing the disease.
 In accordance with the present invention, it has been determined
that EDG1 protein is primarily localized in the nucleus of normal
mammary epithelial cells. It has also been determined that EDG1
protein, if present, localizes predominantly in the cytoplasm in
cancerous mammary epithelial cells and that the extent of cytoplasmic
localization correlates with the stage of the cancer, i.e., more
EDG1 protein localizes in cytoplasm of cells derived from advanced
carcinomas. Thus, it is preferred that the antibody-based detection
methods employ cell tissue sections, since the information obtained
from such samples permit not only detection of cancerous cells,
but also an assessment of the grade of the tumor that is detected.
 Methods of Inhibiting Proliferation of Cancer Cells
 The EDG1 polynucleotides and proteins may also be used to
block the growth or decrease the proliferation of hormone responsive
cancer cells derived from breast tissue, prostate tissue, ovarian
tissue, uterine tissue and testicular tissue. The polypeptides may
be used to decrease proliferation of both hormone sensitive and
hormone insensitive cancer cells that are derived from these tissues,
including estrogen receptor positive and estrogen receptor negative
breast cancer cells. The EDG1 polynucleotides and proteins may be
used to block proliferation of these cancer cells in vitro or in
vivo. The EDG1 polynucleotides and proteins may also be used to
reduce or inhibit proliferation of colon cancer cells. The method
involves increasing the levels of the EDG1 protein in the cancerous
 Inhibiting Proliferation with EDG1 Polynucleotides and Oligonucleotides
 In one embodiment, polynucleotides encoding the EDG1 protein
or a functional equivalent thereof are introduced into such cells
to permit expression or overexpression of the EDG1 protein. Viral
or plasmid vectors may be used to deliver the polynucleotide to
 Levels of EDG1 may be increased in cancer cells by introducing
a DNA fragment comprising an EDG1 polynucleotide and a promoter
into the cell and expressing the EDG1 protein. Preferably, the promoter,
which is operably linked to the EDG1 polynucleotide is a tissue
specific promoter. The DNA fragment may be incorporated into a viral
vector or into a liposome which, preferably, further comprises a
molecule which targets the liposome to the cancer cell. Alternatively,
levels of EDG1 are increased in the target cancer cell by delivering
EDG1 into the cell via a liposome.
 Viral Vector
 Examples of known viral vectors are recombinant viruses
which are generally based on several virus classes including poxviruses,
herpesviruses, adenoviruses, parvoviruses and retroviruses. Such
recombinant viruses generally comprise an exogenous gene under control
of a promoter which is able to cause expression of the exogenous
gene in vector-infected host cells. Recombinant viruses which can
be used to transfect cells are mentioned and cited for example in
a review by Mackett, Smith and Moss (1994) J Virol 49(3): 857-864.
 Preferably, the virus vector is a defective adenovirus which
has the exogenous gene inserted into its genome. The term "defective
adenovirus" refers to an adenovirus incapable of autonomously
replicating in the target cell. Generally, the genome of the defective
adenovirus lacks the sequences necessary for the replication of
the virus in the infected cell. Such sequences are partially or,
preferably, completely, removed from the genome. To be able to infect
target cells, the defective virus must contain sufficient sequences
from the original genome to permit encapsulation of the viral particles
during in vitro preparation of the construct.
 Preferably, the adenovirus is of a serotype which is not
pathogenic for man. Such serotypes include type 2 and 5 adenoviruses
(Ad 2 or Ad 5). In the case of the Ad 5 adenoviruses, the sequences
necessary for the replication are the E1A and E1B regions. Methods
for preparing adenovirus vectors are described in U.S. Pat. No.
5,932,210, which issued in August, 1999 to Gregory et al., U.S.
Pat. No. 5,985,846 which issued in November, 1999 to Kochanek et
al, and U.S. Pat. No. 6,033,908 which issued in March, 2000, to
Bout et al.
 More preferably, the virus vector is an immunologically
inert adenovirus. As used herein the term "immunologically
inert" means the viral vector does not encode viral proteins
that activate cellular and humoral host immune responses. Methods
for preparing immunologically inert adenoviruses are described in
Parks et al., Proc Natl Acad Sci USA 1996; 93(24) 13565-70; Leiber,
A. et al., J. Virol. 1996; 70(12) 8944-60; Hardy s., et al, J Virol.
1997, 71(3): 1842-9; and Morsy et al, Proc. Natl. Acad. Sci. USA
1998. 95: 7866-71, all of which are specifically incorporated herein
by reference. Such methods involve Cre-loxP recombination. In vitro,
Cre-loxP recombination is particularly adaptable to preparation
of recombinant adenovirus and offers a method for removing unwanted
viral nucleotide sequences. Replication deficient recombinant adenovirus
lacks the E1 coding sequences necessary for viral replication. This
function is provided by 293 cells, a human embryonic kidney cell
line transformed by adenovirus type. First generation adenoviruses
are generated by co-transfecting 293 cells with a helper virus and
a shuttle plasmid containing the foreign gene of interest. This
results in the packaging of virus that replicates both the foreign
gene and numerous viral proteins. More recently, 293 cells expressing
Cre recombinase, and helper virus containing essential viral sequences
and with a packaging signal flanked by loxP sites, have been developed
(See Parks et al.) In this system, the helper virus supplies all
of the necessary signals for replication and packaging in trans,
but is not packaged due to excision of essential sequences flanked
by loxP. When 293-Cre cells are co-transfected with this helper
virus, and a shuttle plasmid (pRP1001) containing the packaging
signal, nonsense "filler DNA", and the foreign gene, only
an adenovirus containing filler DNA and the foreign gene is packaged
(LoxAv). This results in a viral recombinant that retains the ability
to infect target cells and synthesize the foreign gene, but does
not produce viral proteins, for targeting cancer cells.
 Methods for targeting vectors to cancer cells are described
in Nakanishi T, Tamai I, Takaki A, Tsuji A. (2000) Cancer cell-targeted
drug delivery utilizing oligopeptide transport activity. Int. J.
Cancer. 88: 274-280, and Poul M A, Becerril B, Nielsen U B, Morisson
P, Marks J D. (2000) Selection of tumor-specific internalizing human
antibodies from phage libraries. J. Mol. Biol. 301: 1149-1161, both
of which are incorporated herein in their entirety. Methods for
delivering isolated oligonucleotides and polynucleotides to cells,
including the nucleus of cells, are described in Lebedeva I, Benimetskaya
L, Stein C A, Vilenchik M. (2000) Cellular delivery of antisense
oligonucleotides. Eur. J. Pharm. Biopharm. 50: 101-119. Review.,
and Fisher K D, Ulbrich K, Subr V, Ward C M, Mautner V, Blakey D,
Seymour L W. (2000) A versatile system for receptor-mediated gene
delivery permits increased entry of DNA into target cells, enhanced
delivery to the nucleus and elevated rates of transgene expression.
Gene. Ther. 7: 1337-1343.
 In a further embodiment an expression construct comprising
the polynucleotide may be entrapped in a liposome. Liposomes are
vesicular structures characterized by a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the formation
of closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat (1991) Targeting of liposomes
to hepatocytes. Targeted Diagn. Ther 4: 87-103). Also contemplated
are lipofectamine-DNA complexes.
 Inhibiting Proliferation of Cancer Cells with EDG1 Protein
and Biologically Active Equivalents Thereof
 Proliferation of cancer cells, particularly breast cancer
cells, may also be accomplished introducing an EDG1 protein or a
biologically active oligonucleotide or polynucleotide derived therefrom
into the cancer cell. A variety of methods exist for introducing
proteins and polypeptides into cells. Such methods include, but
are not limited to, "protein transduction" or "protein
therapy" as described in publications by Nagahara et al. (Nagahara,
et al., 1998, Nat Med. 4: 1449-52.) and in publications from the
laboratory of Dowdy (Nagahara, et al., 1998, Nat Med. 4: 1449-52.;
Schwarze, et al., 1999, Science, 285:1569-72.; Vocero-Akbani, et
al., 2000, Methods Enzymol, 322:508-21; Ho, et al., 2001, Cancer
Res, 61:474-7.; Vocero-Akbani, et al., 2001, Methods Enzymol, 332:36-49;
Snyder and Dowdy, 2001, Curr Opin Mol Ther, 3:147-52.; Becker-Hapak,
et al., 2001, Methods, 24:247-56.), publications which are incorporated
herein by reference.
 In one embodiment an eleven amino acid sequence, the "protein
transduction domain" (PTD), from the human immunodeficiency
virus TAT protein (Green and Loewenstein, 1988, Cell, 55:1179-88.;
Frankel and Pabo, 1988, Cell, 55:1189-93.) is fused to the wild-type
EDG1 protein. The purified protein is then put in contact with the
surface of cells and the cells take up the wild-type EDG1 protein
which functions to inhibit or suppress growth of that cell. In the
case where it is desired to introduce the wild-type EDG1 protein
containing the fused PTD into cells comprising a tumor in a human
or animal, the protein is administered to the human by a variety
of methods. Preferably, the protein is administered by injection
(e.g., intravenously) or by inhalation in an aerosol.
 EDG1 proteins that contain the fused PTD are preferably
made by fusing the DNA sequence encoding the EDG1 protein or a functional
equivalent thereof with the DNA sequence encoding the PTD. The resulting
EDG1-PTD fusion gene is preferably incorporated into a vector, for
example a plasmid or viral vector, that facilitates introduction
of the fusion gene into a organism and expression of the gene at
high levels in the organism such that large amounts of the fusion
protein are made therein. One such organism in which the vector
containing the fusion gene can be expressed is a bacterium, preferably
Escherichia coli. Other organisms are also commonly used by those
skilled in the art. After the fusion protein is expressed at a high
level in any of these organisms, the fusion protein is purified
from the organism using protein purification techniques well known
to those skilled in the art.
 The present invention also provides a method for inhibiting
the transcriptional activity of estrogen-liganded ER.alpha. in cells,
particularly in breast cancer cells. Such method comprises increasing
levels of the EDG1 protein in such cells.
 The invention may be better understood by reference to the
following examples, which serve to illustrate but not to limit the
 Tissue culture and transfections
 Breast epithelial cells (MCF7, MCF10A, MDA-MB-231 and T47D)
and PA317 amphotropic packaging cells were obtained from ATCC and
maintained according to their recommended protocols. HBL100 cells
were provided by Dr. David L. McCormick (I.I.T. Research Institute,
Chicago, Ill.) and were maintained in Minimum Essential Medium (MEM)
plus phenol red supplemented with 5% heat-inactivated fetal calf
serum. CHO cells were maintained and transfected as previously described
 The EDG1 clone, pAD-GAL4-2.1-EDG1 , obtained from yeast
two hybrid screening contains coding sequence cloned in frame with
the activation domain of GAL4 in the pAD-GAL4-2.1 phagemid vector
(Stratagene, La Jolla, Calif.). The EDG1 cDNA clone was released
by NcoI/XbaI digestion, blunted and inserted into SalI/SmaI-digested
pCMV5 vector to make pCMV5-EDG1 . The NcoI/XbaI blunted EDG1 fragment
was inserted into BamHI-digested and blunted pBPSTR1 retroviral
vector in the sense or antisense direction to make pBPSTR1-EDG1
or pBPSTR1-EDG1.sub.AS respectively. pGEX2T-EDG1 , which encodes
full-length EDG1 in frame with glutathione-S-transferase (GST) was
constructed by inserting NcoI/XbaI blunted EDG1 fragment into BamHI
digested and blunted pGEX2T (Pharmacia, Piscataway, N.J.). pEGFP-EDG1
, which encodes full length EDG1 in frame with the coding sequence
for Green Fluorescent Protein (GFP), was constructed by inserting
NcoI/XbaI blunted EDG1 fragment into HindIII-digested and blunted
pEGFP-C3 vector (Clonetech).
 To make pRFP-67LR, 67LR reading frame was generated by PCR
using the yeast two hybrid clone, pAD-GAL4-2.1-67LR, containing
the complete coding sequence of 67LR in frame with GAL4 in the pAD-GAL4-2.1
phagemid vector, as template. Reactions were performed using Platinum
Pfx DNA polymerase (GIBCO) according to the manufacturer's recommendations.
The following PCR primers were used:
2 67LRf: 5' ACACAGGATCCGAATTCATGTCCGGAGCCCTTGATGTC-3', SEQ. ID
NO.: 6 67LRr: 5'-ACACAGGATCCAGTCGACTAAGACCAGTCAGTGGTTGCTCCT-3- ',
SEQ. ID NO.: 7.
 The PCR fragment was purified, digested with BamHI, and
cloned into BglII-digested pRFP-C1 vector.
 Yeast Two Hybrid Screenings
 The yeast two hybrid screenings used to identify ER.alpha.-
and EDG1-interacting clones were described previously (Montano M
M, Ekena K, Chang W C, Katzenellenbogen B S (1999) An estrogen receptor
selective corepressor that potentiates the effectiveness of antiestrogens
and represses the activity of estrogens, Proc. Natl. Acad. Sci.
 In Vitro Translation and Protein-Protein Interaction Assays
 In vitro transcription and translation of ER.alpha., EDG1
or 67LR and p27/BBP were performed using the Promega TNT kit (Madison,
Wis.) according to the manufacturer's recommendation. GST-pull down
assays were previously described (Montano M M, Ekena K, Chang W
C, Katzenellenbogen B S (1999) An estrogen receptor selective corepressor
that potentiates the effectiveness of antiestrogens and represses
the activity of estrogens, Proc. Natl. Acad. Sci. 96: 6947-6952).
 Northern Blot Analysis
 RNA was extracted from breast epithelial cells using Trizol
(GIBCO) and was subjected to Northern Analyses as described previously
(Montano M M, Jaiswal A, Katzenellenbogen B S. (1998) Transcriptional
regulation of the human quinone reductase gene by antiestrogen-liganded
estrogen receptor .alpha. estrogen receptor .beta. via the electrophile/antioxidan-
t response element. Journal of Biological Chemistry. 273: 25443-25449).
 Retroviral-Mediated Transfection
 Retroviruses were made by transfecting PA317 cells with
the pBPSTR1 plasmid alone or pBPSTR1 containing EDG1 in the sense
or antisense orientation. Breast epithelial cell lines were infected
with retrovirus-containing supernatants in the presence or absence
of 3 ug/ml tetracycline. When tetracycline was added, expression
of the viral gene was inhibited. Changes in EDG1 mRNA were verified
by harvesting RNA from infected cells for Northern blot analyses
or by immufluorescence staining.
 Immunofluoresence Staining of Breast Cells and Tissues
 Breast tissue samples were fixed in formalin, embedded in
paraffin, and sectioned at 5 micron thickness. To unmask epitopes
we used heat-induced antigen retrieval technique using 10 mM Tris.
After blocking with 10% normal goat serum, sections were incubated
with EDG1 peptide 152-171) polyclonal rabbit antibody and goat,
anti-rabbit IgG Alexa 488 fluorescence secondary antibody. As a
negative control duplicate sections were immunostained with nonspecific
 Cells grown on coverslips were fixed in paraformaldehyde.
After blocking with serum, samples were incubated with EDG1 primary
and seconday antibody as described above. To detect 67LR cells were
immunostained using 67LR IgG monoclonal mouse antibody (Lab Vision)
and goat, anti-mouse Alexa 594 secondary antibody
 Anchorage Independent Growth
 Four days after infection cell were detached and suspended
at a concentration of 1.times.10.sup.4 in medium containing 0.3%
agar and then plated in a 6-well plate precoated with 0.9% agar
base layer. At 24 h and 21 days after plating colonies larger than
50 .mu.m were counted.
Effects of EDG1 on (E.sub.2)-Liganded ER.alpha. Transcriptional
 Estrogen Down-Regulated Gene 1 (EDG1) was identified by
yeast two hybrid screenings for ER interacting proteins in breast
epithelial cells. Because EDG1 interacted with Estradiol (E.sub.2)-liganded
ER.alpha. (FIG. 2A) we determined if EDG1 would have an effect on
the transcriptional activity of E.sub.2-liganded ER.alpha.. We observed
down-regulation of ER.alpha., Progesterone Receptor .beta. (PR.beta.)
and Retinoic Acid Receptor .alpha. (RAR.alpha.) transcriptional
activity in the presence of increasing amounts of expression vector
for EDG1 (FIG. 2B). EDG1 did not inhibit the transcriptional activity
of another transcriptional activator, VP16. Thus the effects of
EDG1 on ER.alpha. transcriptional activity cannot be attributed
to general breakdown of transcription. Fluorescence studies show
that transfected EDG1 localizes to the nucleus (FIG. 3C).
Detecting Cancerous Breast Epithelial Cells with Anti-EDG1 Antibody
 EDG1 protein expression in breast tumor samples and adjacent
normal breast tissues from 16 subjects was examined using immuncytochemical
techniques. Results from representative samples are shown in FIG.
4B. As expected EDG1 expression was observed in the nuclei of endothelial
blood vessels (Patient III, row 4, indicated by arrow). High levels
of EDG1 protein was also detected in 15 of 16 normal breast tissue
samples, specifically in the nuclei of epithelial duct cells (Patient
I, II, and III, row 4). EDG1 protein was present in the epithelial
cell nuclei of 1 of 3 ductal carcinoma samples in situ (Patient
III, row 2) and the epithelial cell nuclei of a Bloom-Richardson
Grade 1 highly differentiated mucinous infiltrating carcinoma (data
not shown). In 11 out of 12 samples of poorly differentiated Grade
II infiltrating ductal carcinoma (Patient I and II, row 2), there
was no EDG1 protein levels in the nucleus and low levels of EDG1
protein in the cytoplasm of the cancerous cells. Thus in addition
to differences in levels and spatial expression of EDG1 protein,
there are differences in intracellular localization of EDG1 protein
in normal breast and breast cancer tissues
Inhibiting Anchorage-Independent Growth with a Polynucleotide Encoding
 Anchorage-independent growth is a necessary requirement
for tumor growth and is a well-established in vitro assay for the
malignantly transformed cellular phenotype. Soft agar colony formation,
a measure of anchorage-independent growth, was examined in control,
MCF7-EDG1 and MCF7-EDG1.sub.AS cells. There is a 72% decrease in
colony formation as a result of increased EDG1 expression, while
increased colony formation was observed in MCF7-EDG1 AS cells (FIG.
Inhibiting Proliferation of Estrogen-Receptor Negative Mammary
Epithelial Cells with a Polynucleotide Encoding EDG1 Protein
 It was then determined if the growth inhibitory effects
of EDG1 were dependent on ER.alpha. status and inhibition of ER.alpha.
transcriptional activity. EDG1 and EDG1.sub.AS retroviruses were
infected into other breast epithelial cell lines that express very
low levels of ER.alpha. and ER.beta. protein (MCF10A), or do not
express the ER.alpha. but express ER.beta. protein (MDA-MB-231).
Decreased expression of EDG1 (0.42.times.) in MCF10A after infection
with EDG1.sub.AS retroviruses is associated with 4-5-fold increase
in proliferation while a slight increase in EDG1 expression (1.8.times.)
inhibited proliferation markedly (FIG. 4E). No significant effects
on proliferation of MDA-MD-231 cells, which already expresses very
low endogenous levels of EDG1, was evident after infection with
EDG1 .sub.AS retroviruses. However after infection with EDG1 retroviruses
we saw a 64% decrease in proliferation (FIG. 4E). These findings
suggest that some of the growth inhibitory effects of EDG1 may occur
independent of ER.alpha. levels and the inhibition of ER.alpha.
transcriptional activity. The importance of these findings is underscored
by the fact that although the growth of some estrogen receptor (ER.alpha.)
positive breast cancers can initially be hormonally manipulated,
all will eventually escape hormonal control. Monica, does this mean
that EDG1 can be used to treat estrogen receptor negative breast
cancers as well as estrogen receptor positive breast cancer.
 It was also observed an increase in lipid vacoule formation,
a measure of breast epithelial cell differentiation, in MCF10A cells
infected with EDG1 retroviruses (FIG. 3E).